Alt a 1 Promotes Allergic Asthma In Vivo Through TLR4-Alveolar Macrophages

The mold Alternaria alternata is one of the main sources of asthma exacerbation, being its major allergen, Alt a 1, indispensable for its development. The main objective of this work was to answer two main questions: 1) can Alt a 1 by itself (without any other context) induce an asthmatic profile in vivo?; and 2) Which molecular mechanisms take place during this phenomenon? To answer both questions, we have developed a mouse model of allergic asthma using only Alt a 1 for mice sensitization. We also made use of in-vitro cellular models and computational studies to support some aspects of our hypothesis. Our results showed that Alt a 1 can induce an asthmatic phenotype, promoting tissue remodeling and infiltration of CD45+ cells, especially eosinophils and macrophages (Siglec F+ and F4/80+). Also, we have found that Alt a 1 sensitization is mediated by the TLR4-macrophage axis.

INTRODUCTION
Asthma is characterized by reversible airway obstruction as the result of predominantly type-2 (T2) driven airway inflammation and pulmonary remodeling. The development of asthma usually starts from repeated environmental allergen exposure (1). Fungal spores constitute the largest proportion of airborne particles, although the majority of mold sensitization research performed points to a few fungal genera to be relevant in airway diseases (2). Alternaria alternata is one of the most common aeroallergens sources (3). It has been long described as an outdoor and indoor mold, and a clear risk factor for asthmatic people when spores reach their highest levels in the air during late summer and/ or early autumn period (4,5). Thus, mouse models have been widely used to characterize the ability of Alternaria to induce T2 immune responses (3,6).
Alt a 1 is the only major allergen related to Alternaria-induced asthma, being a marker of primary sensitization and chronic asthma (7). Although the biological function of Alt a 1 remains unknown, it seems to show a role in plant pathogenesis (8,9). Alt a 1 is a small protein (~15 kDa) mainly detected in spores (10) from where it is released in large quantities as a tetramer at pH around 5.0 -6.5, carrying a flavonol molecule as a ligand (11). Its tetrameric form allows its recognition by SLC22A17, a receptor in bronchial epithelial cells, inducing the production of alarmins such as IL-33 and IL-25 (12). In contrast, Garrido-Arandia et al. also described that this interaction does not affect the permeability of the epithelium (12), and therefore, it cannot be responsible for the entry of Alt a 1 through the epithelium. Thus, there must be another mechanism for the allergen to pass through the epithelium and activate the inflammatory response. Lastly, Alt a 1 has been described to be able to induce innate immunity, depending mainly on TLR4 (6).
Little is known about the mechanisms underlying Alt a 1 sensitization, and many questions remain unanswered, such as the relationship between asthma and Alt a 1, or whether some other component of the spores is necessary helping to induce the T2 response. Similarly, we do not know how Alt a 1 can pass through the epithelium and be presented by the antigen-presenting cells (APC) beneath it, inducing the adaptive response.
The goal of this study has been the characterization of the immunological activity of Alt a 1 in vivo by developing a mouse model of allergic asthma. Furthermore, in view of the limited information regarding molecular mechanisms underlying the high prevalence of Alt a 1 sensitization, we also intended to focus on the description of the first interaction between Alt a 1 and the airway environment, extending the interest to the role of alveolar macrophages (AM). In this study, we investigated the innate immunostimulatory activities of Alt a 1 in the lung as well as in a human epithelial bronchial model. We found that Alt a 1 shows a potent cytokine-and chemokine-inducing activity, mostly dependent on Toll-like receptor signaling pathways. Moreover, evidence is shown for a significant interaction between TLR4 and Alt a 1, which could be responsible for TLR dimerization and the activation of the inflammatory cascade.

Mouse Model of Allergic Asthma
Female C57BL/6J mice were obtained from Charles River breeding colony (France); housed in the animal care facilities at IIS-Fundacioń Jimeńez Dıáz (Madrid, Spain) under standard laboratory conditions. All animal protocols and procedures were approved by the Institutional Animal Care and Use Committee from Community of Madrid (Ref. PROEX 392/15) and were conducted in compliance with current legislation (European Union Directive 2010/63/EU).

Humoral Response
The detection of Alt a 1-specific immunoglobulin (Ig) in sera was performed by ELISA and the proper HRP-conjugated antibodies when it was required. Sera were diluted 1:5 for IgE and 1:50 for both IgG bindings (overnight; 4°C). Optical density was determined by measuring the absorbance at 450 nm.

Bronchoalveolar Lavage (BAL) Cell Analysis
Once the trachea was exposed and incised, a needle (1.2 x 40 mm) was inserted into the trachea and BAL was harvested by rinsing the lungs twice with 1 ml of sterile PBS. Total cell counts were determined with a hemocytometer and the cellular profile of BAL was obtained by flow cytometry using specific antibodies as indicated below. Briefly, after fixation with % PBS-formaldehyde (PFA; 5 min, 25°C), cells were stained with the specific fluorochrome-conjugated antibodies for 1h at RT. After washing, samples from Alt a 1-sensitized mice were analyzed using a MA900 Multi-Application Cell Sorter (Sony Biotechnology, USA). Unstained controls and single stained controls were processed to correct compensation.

Measurement of Lung Function
Lung resistance was measured using a Numiotec ventilator system (Numio Technologies, SL, Madrid, Spain) following provider's instructions. To this end, inspiration/expiration plots were registered for each mouse for 10 min (3 cycles). Control measurements were obtained over a period of 5 min directly after administration of rocuronium. The average plot was registered for every mouse and the plot represented is the average for each group treated. The maximum point of volume/ pressure represents the dynamic compliance and the space between the lower line (inspiration) and upper line (expiration), which is directly related to lung expansion.

Characterization of Lung Cell Infiltrates
To obtain single-cell suspensions, lungs (n = 4/group) were cut into small pieces with blades and digested for 30 min at 37°C in PBS containing 0.1% v/v of BSA (Gibco, Thermo Fisher), 1 mg/ ml collagenase A (Roche, Switzerland) and 0.1 mg/ml DNase I (Roche). After, cell aggregates were dissociated, and cells were fixed with 1% PFA for 5 min at RT. Cell sorting was performed on a MA900 (Sony) at a rate allowing minimum 90% of efficiency to recover CD45+ cells. Leucocyte population was subsequently characterized using specific antibodies listed in Supplementary Table 1.

Gene Expression Profiling
Lungs were homogenized and lysed with GIT extraction buffer (pH 7; 4 M guanidine isothiocyanate, Sigma-Aldrich; 25 mM sodium citrate, Sigma-Aldrich; 0.5% sarcosyl, Sigma-Aldrich; 0.1 M 2-mercaptoethanol, Carl Roth, Germany). RNA was purified by phenol:chloroform extraction and precipitated with ethanol. cDNA was obtained and a pool of 5 mice/group was analyzed by qPCR using Bio-Rad's PrimePCR Pathways (Asthma & Allergy, M384; Hercules, CA, USA), following the provider's instructions. To identify the differentially expressed genes between the Alt a 1 group and the control group, the GeneStudy_1.0.030.1023 software was applied.

Lung Lysates for Western Blotting
Snap-frozen lung tissues were homogenized in RIPA buffer containing 20 mM Tris-HCl (pH 8), 150 mM NaCl, 0.1% SDS, 1% Triton X-100 and EDTA-free protease inhibitor cocktail (Roche). After that, samples were centrifuged for 10 min (4°C) at 10.000 g and supernatants were collected and quantified by Bradford method. Lung lysates were separated by SDS-PAGE (15% acrylamide) and the ORMDL-3 and caspase-1 immunodetections were assured for the use of specific antibodies listed in Supplementary Table 1, and the subsequent use of HRP-conjugated anti-IgG antibodies. Antitubulin was included to show equal loading and transference of lung protein extracts. The quantification of the bands was carried out by optical densitometry and analyzed using the ImageJ digital imaging processing software (ImageJ 1.53a, National Institutes of Health, Bethesda, MD, USA). The expression of each protein was normalized with b-tubulin signal.

Co-Localization of Alt a 1 With Endosome Markers
To characterize the Alt a 1 traffic, non-polarized Calu-3 cells (ATCC; HTB-54; USA) were grown on coverslips until reached a 70% of confluency. Then, they were incubated with Alt a 1 at 37°C, 5%CO2 for different times: 5 min for early endosome assay, and 30 min for recycling endosome assays. After incubation, cells were washed, fixed with 4% PFA for 10 min and permeabilized with PBS-Triton X100. Cells were blocked and then they were incubated with anti-Alt a 1 antibody and the proper anti-endosome marker (all antibodies listed in Supplementary Table 1) for 1h at RT. After using Alexaconjugated secondary antibodies, cells were stained with DAPI and mounted with ProLong Gold over slides. Images were obtained with a Zeiss LSM 880 confocal microscope, using 405, 488 and 633 laser excitations and 63X amplification.
The recycling pathway was inhibited by adding 100 mM endosidin-2 (ES-2; Sigma-Aldrich) 1h prior to adding Alt a 1 to Calu-3 monolayers. Alt a 1 accumulation was evaluated by immunofluorescence following the earlier described protocol.

Pull-Down Assays
Silica nanospheres (80 nm carboxyl; nanoComposix, California, USA) were conjugated with TLR 4 (Peprotech, New Jersey, USA) in 50 mM MES buffer pH 4.5 containing 0.01% Tween-20 overnight at RT. The functionalized nanoparticles were blocked with 50 mM Tris-HCl pH 8 and casein blocking buffer for overnight at 4°C. For pull-down assays, nanoparticles were incubated with Alt a 1 (0.1 mg/mL) or Complex (0.1 mg/ml) for 2h at room temperature. After centrifugation, pellets were extensively washed and resuspended in denaturing Laemmli buffer. Samples were separated into a 15% SDS-PAGE and the biding of Alt a 1 or Complex was confirmed by western blot using a specific anti-Alt a 1 antibody. Negative controls of nonfunctionalized nanoparticles incubated with Alt a 1 or functionalized nanoparticles without Alt a 1 were included.

Lung Immunofluorescence
Intact lung halves (neither treated nor paraffined) from healthy C57BL6/J mice were incubated with Alt a 1 (25 mg) or saline (PBS) for 10 min at 37°C in RPMI 1640 medium (Invitrogen, USA) containing 2 mM L-glutamine, 25 mM HEPES, 10% heatinactivated fetal bovine serum, and 1% Pen-Strep. Then, lungs were fixed with 4% PFA for 16 h and cut into thin slices. After blocking, lung slices were stained with specific antibodies (listed in Supplementary Table 1) for 1h at RT, followed by secondary antibody conjugated with Alexa Fluor Dyes. Nuclei were stained with DAPI, and negative controls of anti IgG-Alexa antibodies were included.

THP1-XBlue-CD14 ™
The transfected cell line THP1-XBlue-CD14 ™ (In vivogen, France) derived from the human monocytic THP-1 cell line was used according to the manufacturer's instructions. Briefly, 1.10 (6) cells/ml were stimulated with Alt a 1 or Complex (5 mg/ ml) for 16 h, and, the NF-kB pathway activation was monitored by the detection of the secreted embryonic alkaline phosphatase (SEAP) in the supernatants. For the inhibition tests, cells were incubated with anti-TLR4 antibodies (5 mg/ml; Invitrogen) or an inhibitor of myeloid differentiation protein 2 (10 mM; MedChemExpress, USA) for 15 min prior to the stimulation. In addition, the presence of TNFa and IFNg was detected in the supernatants from Alt a 1-induced THP1 cells using ELISA kits (ImmunoTools GmbH, Germany), and the results were normalized by the non-stimulated values.
To obtain human macrophages-like cells, THP-1 cells were treated with a final concentration of 100 nM Phorbol 12-Myristate 13-Acetate (PMA; Sigma Aldrich, USA) in RPMI medium for 48h. THP1-derived macrophages were cultured in coverslips and, once they reach a stable morphological state, cells were incubated with Alt a 1 for 2 min at 37°C. Cells were immediately fixed with 4%PFA and TLR4 and Alt a 1-Alexa 647 antibodies were used for an immunofluorescence assay. Nuclei were stained with DAPI and a negative control of secondary antibodies was included. Images were obtained with a Zeiss LSM 880 confocal microscope, using 405, 488 and 633nm laser excitations.

Computational Analysis
Structural comparison between Alt a 1 and myeloid differentiation factor 2 (MD2) was compute using three structural alignment methods implemented on the protein data bank website: FATCAT (13), CE (14) and TM-Align (15). Moreover, it also was analyzed using MMLigner (16), a statistical inference of protein structural alignments and the presence of concepts in the Alt a 1 structure was identified using Procodic (17). The PDB entries 3V0R (18) and 3FXI (19) were used for Alt a 1 and MD2 structure respectively. In order to analyze the interaction between TLR4 and Alt a 1, the initial geometry was obtained with blind docking calculations using ZDOCK (20), and ColabFold (21). Among the models obtained, the initial geometry was chosen based on the similarity of the Alt a 1 localization with respect to MD2 in the TLR4-MD2 crystal structure (PDB: 3FXI).
Alt a 1-TLR4 molecular system and Alt a 1-TLR4 tetramer system was explored with all-atom 50 and 100 ns Molecular Dynamics (MD) simulations using the CHARMM3.1 force field and the multicore CUDA version of NAMD 2.13 in the Tesla V100 GPU of the highperformance computing CBGP. The system was prepared with CHARMM-GUI (22,23).Periodic solvation boxes with 14 Å spacing in all dimensions and TIP3P (24) water model were used and Na+ and Cl-ions added to counter total charges and set 0.150 M salt concentration. Initial geometries were minimized at 5000 conjugate-gradient optimization steps and water was then equilibrated at 298 K and 1 atm for 100 ps at 2 fs time steps. Production runs were performed during 100 ns simulation time at 2 fs timesteps in the NPT ensemble at 1 atm and 298 K with Langevin dynamics for T control and Nose-Hoover Langevin piston method for P control. Trajectories were processed and analyzed with VMD 1.9.3 (25).The dissociation constant of the TLR4-Alt a 1 and TLR4-MD2 complexes were calculated using Prodigy web server (26,27).
Molecular graphics were prepared and rendered with UCSF Chimera v1.16 (28). Protein Data Bank the was also analyzed using MMLigner (16). Structures, Alt a 1-TLR4 and MD2-TLR4 tetramer systems were explored with all-atom 100 ns Molecular Dynamics (MD) simulations using the CHARMM 3.1 force field and the multicore CUDA version of NAMD 2.13 in the Tesla V100 GPU of the high-performance computing CBGP. Periodic solvation boxes and water model were used, and Na+ and Clions were added to counter total charges and set 0.150 M salt concentration. Initial geometries were minimized at 5000 conjugate-gradient optimization steps and water was then equilibrated at 298 K and 1 atm for 100 ps at 2 fs timesteps. Production runs were performed during 100 ns simulation time at 2 fs timesteps (50 million steps per simulation) in the NPT ensemble at 1 atm and 298 K with Langevin dynamics for T control and Nos-Hoover Langevin piston method for P control (28).

Statistical Analyses
Statistically significant differences were analyzed by Prism9 (GraphPad Software Inc., La Jolla, CA, USA) using Kruskal-Wallis test with Dunn's correction for multiple comparisons and P-values < 0.05 were considered significant.

Alt a 1 Can Trigger Allergic Asthma in a Mouse Model
Intranasal exposure of mice to Alt a 1 ( Figure 1A), in the presence (Complex) or absence (Alt a 1) of its ligand (a quercetin-like molecule), elicited allergic airway inflammation. No differences could be observed as a result of the presence of the ligand. Alt a 1 sensitization was confirmed by measuring specific humoral response (Figures 1B-D). This response was only induced by the presence of the allergen and not because of putative environmental pollution, as suggested by the BSA-treated group lacking specific IgE ( Figure 1E). Moreover, we observed differences in the respiratory capacity between mice with Alt a 1-induced asthma and controls ( Figure 1F), the first ones also showing severe phenotypic changes in the lung tissue such as sub-epithelial fibrosis, cell infiltration and goblet cell hyperplasia ( Figure 1G).
The asthmatic phenotype observed can only be due to the presence of Alt a 1. When another allergen was used as OVA following the same pattern as for the Alternaria allergen, no humoral response, no cellular infiltration in the BALF and no alveolar remodeling could be seen ( Figures 3A-D).

Alt a 1-Induced Asthma Is Characterized by an Increase in the Expression of Asthma Markers
The asthmatic phenotype of Alt a 1-sensitized mice was characterized by analyzing the expression of asthma markers such as ORMDL-3 (29) and caspase-1 (30). The presence of both markers was increased in the lungs of asthmatic mice after Alt a 1-sensitization as can be seen in Figure 4A. The characterization of the asthmatic profile was completed by transcriptomic analysis study comparing asthmatic versus healthy mice. In this way, 42 genes were found up-regulated (> 4-fold), most of which are related to eosinophilic asthma. The majority of those genes (n=27) corresponds to T2 cytokines and alarmins (i.e., IL25, IL33, IL13); chemokines (i.e., IL5); their receptors (i.e. IL4ra, IL5ra) ( Figure 4B); or genes related to lung impairment (i.e., Muc5a, Ccll2 or Ccll3) and mast cell functions (i.e., Fcer1a, Tpsb2 or Kitl) ( Figure 4C).

Alt a 1 Is Recognized and Directed to the Recycling Pathway
To study how Alt a 1 can pass through the epithelial barrier, an in vitro bronchial epithelium model was developed using the human Calu-3 cell line. According to previous data, Alt a 1 can interact with epithelial cells through the SLC22A17 receptor, however the percentage of transported protein is too low to explain the recognition by APCs11. Studying the Alt a 1 internalization into epithelial cells, no co-localization was observed between Alt a 1 and EEA1 (early endosome antigen-1), an early endosome marker, suggesting that protein follows an EEA1-independent pathway. In contrast, Alt a 1 co-localized with Rab 11, a marker of the endosomal-recycling pathway ( Figure 5A). This result was confirmed through the inhibition with endosidin-2 (ES-2), an exocytosis and endosomal recycling inhibitor ( Figure 5B). Thus, Alt a 1 could be recycled following the same route as in the case of lipocalin-2, another ligand of SLC22A17 ( Figure 5C) (31).

Alt a 1 Induces NF-kB Signaling by TLR4 Pathway
Since the recycling of Alt a 1 might be partially preventing the apical-to-basal transport of the protein through the epithelial barrier, and considering that the protein does not alter the epithelial permeability in vitro, the mechanism through which Alt a 1 reaches APCs on the basolateral side remains unclear. One possibility is the recognition of Alt a 1 by AM, which could directly introduce the allergen in the alveoli triggering a pro-inflammatory response, as previously described6. Supporting this hypothesis, colocalization of Alt a 1 with TLR4 in mice lung F4/80+ cells ( Figure 6A) as well as in THP1-derived macrophages ( Figure 6B) was observed in immunofluorescence assays. This recognition was also confirmed by pull down-binding assay, using TLR-nanoparticles incubated with Alt a 1 ( Figure 6C). When Alt a 1 was cultivated with a macrophage cell line, THP1-XBlue-CD14+, the interaction between TLR4 and Alt a 1 was inhibited by adding specific antibodies or specific MD2 inhibitor (32) ( Figure 6D).

Alt a 1 Shares Structural Homology With MD2 Being Able to Mimic the Role of the Complex MD2-LPS
Based on these results, the structural similarity between Alt a 1 and MD2 was analyzed. Both are small proteins of 15 and 18.5 KDa respectively with a structure characterized by the presence of beta sheets. Although the scores obtained in the similarity analysis do not reveal that they belong to the same protein family, a structural relationship between both can be observed ( Figure 7A).
To explore whether such structural similarity might be sufficient to complex with TLR4, the interaction between Alt a 1 and TLR4 was explored by means of molecular dynamics simulations using as a reference the interaction between TLR4 and MD2. The results obtained, reveal that the Alt a 1-TLR4 complex remains stable along the simulation (100 ns), as shown by the stabilization of the RMSD values ( Figure 7B). This complex is maintained by the presence of more than 18 hydrogen bonds ( Figure 7C). In addition, the distances between the Glu 586 and the Asp 325, in the C terminal region of the TLR4 ectodomain, between each protein of the homodimer were analyzed to know the behavior along the dynamics simulations. It can be seen how in the case of TLR4 with Alt a 1, the distance between the residues is more similar to the crystallographic structure of TLR-MD2-LPS than in the case of TLR4-MD2, in which a high increase could be observed ( Figure 7D). All these data suggest that Alt a 1 could be (C) Detection of a profile of cytokines in supernatants of BALFs collected from PBS-treated mice (control group) and Alt a 1-treated mice. Data shown are from a twenty minute exposure to X-ray film, using the ImageJ software (version 1.53a) to quantify the signals.
bound with high affinity to TLR4, in absence of MD2, promoting the dimerization of the receptor.

DISCUSSION
Alternaria alternata has been reported as one of the most important molds acting as an allergic asthma inducer (10). The present study provides evidence that Alt a 1, regardless of the ligand, promotes airway allergic inflammation in an asthma mouse model. Alt a 1-sensitization induced an airway type 2 inflammation, marked by up-regulation of genes related to eosinophilia and CD45+ infiltration, mucous hypersecretion, and increased of specific antibodies in serum (sIgG1, sIgG2 and sIgE). Even more, Alt a 1asthmatic mice showed an increase of ORMDL-3 and caspase-1 in the asthmatic mice, both markers related to severe asthma. In the case of ORMDL-3, it has been associated to eosinophil trafficking, their recruitment and degranulation (33,34), although its expression did not influence in the number of T2 cells. Referring to caspase-1, it can induce the T2 cytokines production inducing the asthma exacerbations. Caspase-1-deficient mice showed no alterations in general lung inflammatory parameters, but a marked reduction in eosinophilia (29). Caspase-1 has been widely studied for promoting the secretion of active forms of IL-1 and IL-18 (35), major contributors to asthma pathogenesis in mice. Focusing on the putative Alt a 1mechanism to induce asthma, the role of the epithelium seems not to be an essential step. As previously reported by Garrido-Arandia et al., Alt a 1 cannot alter the barrier integrity, being very limited its transport across the polarized bronchial cell model (less than 10%) (12). These previous studies have also shown that Alt a 1 can interact with the bronchial epithelium via the SLC22A17 receptor in the presence of its ligand, inducing the production of alarmins and pro-inflammatory cytokines. In contrast, this interaction does not seem to induce the Alt a 1 transport to basolateral side, but its recycling, according to the colocalization with Rab11, the most well-established mediator of endosomal recycling (36). The higher accumulation of Alt a 1 inside epithelial cells that had been previously incubated with an exocytosis inhibitor, ES-2, supported these results. This may explain why Alt a 1 is poorly detected in the basolateral side when the cell model was analyzed (6,12). However, Alt a 1 needs to reach the basolateral side, otherwise, there would be no production of specific antibodi es against this a llergen a nd the p athway undertaken should be independent of SLC22A17.
Considering that AM represent the largest population of immune cells resident in the lung in a healthy state, Alt a 1 could be taken in by AM (F4/80+ cells) in lungs from healthy mice, and notably, co-localizing with TLR4 receptor. Previously, Hayes et al. demonstrated that IL-8 and NF-kB induction by Alt a 1 in bronchial epithelial-and embryonic kidney cell culture was mostly dependent upon TLR4 signaling, but also upon TLR2 and the adaptor proteins MyD88 and TIRAP, suggesting a key role of these receptors in Alt a 1-induce innate immune responses (6). In line with this hypothesis, we have described the Alt a 1 ability to interact with TLR4. We further confirmed in an in vitro monocytes culture (THP1) that the Alt a 1-induced NF-kB pathway is mediated by TLR4. The inhibition of Alt a 1induced NF-kB signaling was complete when THP1 cells were previously incubated with TLR4 blockade antibody while the co-adaptor protein MD2 seems to be participating in this TLR4-mediated Alt a 1 recognition. Computational analyses supported this mechanism related to Alt a 1-TLR4 interaction, showing the presence of a stable complex along 100 ns MD simulations, maintained by 39 interchain hydrogen bonds. Moreover, the similarity between the final s t r u c t u r e o f t h e A l t a 1 -T L R 4 c o m p l e x a n d t h e crystallographic TLR4-MD2-LPS, suggests that the presence of Alt a 1 would be able to keep the Ct-region of the extracellular domain of TLR in close contact, allowing dimerization of the intracellular domains and the activation of the TLR pathways.
Alt a 1-mediated facilitation of TLR4 signaling in the presence of very low (or no) LPS exposure may promote the T2 response. Similarly, Alt a 1 probably promotes the development or exacerbation of asthma, possibly due to its ability to interact with TLR4 on alveolar macrophages in the airways. In this case, the ability of Alt a 1 to reconstitute TLR4 signaling and activation, in the absence of ligand, may be of particular importance. Indeed, the structural similarity between MD2 and Alt a 1 would only explain why Alt-1 can interact with TLR4, but not why it can activate the TLR4 activation cascade without ligand. This fact highlights the ability of a protein to activate the immune system towards an asthma phenotype without the need of adjuvant activity or associated ligands as has been described for other proteins. In summary, this study has highlighted two events: first, the description of the ability of Alt a 1 to induce allergic airway inflammation in a mouse model regardless of the presence of a ligand; and, second, the potential role of lung macrophages mediating the Alt a 1-immune responses (Figure 8). Although the interaction of Alt a 1 with TLR4 is unlikely to be of biological importance to the mold, it is tempting to speculate that Alt a 1 plays an essential role in establishing the ideal conditions for the fungus development. The pathophysiological interactions of Alt a 1 with the immune system may be more than a unique oddity.

DATA AVAILABILITY STATEMENT
The original contributions presented in the study are included in the article/Supplementary Materials, further inquiries can be directed to the corresponding author/s.

ETHICS STATEMENT
The animal study was reviewed and approved by Institutional Animal Care and Use Committee from Community of Madrid.


Quorum Sensing Inhibition and Metabolic Intervention of 4-Hydroxycinnamic Acid Against Agrobacterium tumefaciens

The natural product 4-hydroxycinnamic acid (HA) was firstly isolated from the metabolites of Phomopsis liquidambari, one endophytic fungus from Punica granatum leaves. The anti-QS potential of HA was evaluated by β-galactosidase assay and acylated homoserine lactones (AHL) analysis. The MIC of HA was > 1.20 mM. Exposure to HA at sub-MIC concentrations (0.30–0.60 mM) remarkably reduced the β-galactosidase activity and AHL secretion. Transcriptional analysis by qRT-PCR and docking simulation indicated that HA functions as an anti-QS agent by inhibiting the transcriptional levels of traI and traR rather than signal mimicry. The blocked QS lead to suppressed biofilm formation, motilities, and flagella formation after exposure to HA at concentrations ranging from 0.30 to 0.80 mM. The dysfunctional QS also resulted in repressed antioxidant enzymes and intensified oxidative stress. The intensified oxidative stress destroyed membrane integrity, induced energy supply deficiency, resulted in disorder of protein and nuclear acid metabolism, and ultimately weakened pathogenicity of A. tumefaciens. HA may have promising potential for controlling A. tumefaciens.

INTRODUCTION
Agrobacterium tumefaciens is a soil-borne phytopathogen and characterized by its ability to cause crown gall disease, a plant tumor affecting a wide range of plants (Lee et al., 2009). A. tumefaciens has the potential to infect more than 1,000 various species of dicotyledonous plants as well as some species of monocots, and many of them are agronomically important crops (Wang et al., 2018). Crown gall disease is responsible for extensive economic losses to nursery productions of fruits plants, roses, grapevines in many countries (Pulawska, 2010). A. tumefaciens infects plants by incorporation of its transfer-DNA (T-DNA) into genome of plant cells, and eventually results in the uncontrolled formation of tumors on account of the unbalanced auxin and cytokinin and uncontrolled cell division. The transfer of T-DNA is activated once A. tumefaciens detects phenols secreted by the wounded plant cells. Studies have shown that the horizontal transfer of T-DNA is tightly mediated by vir genes (Lang et al., 2013). Therefore, suppressing vir gene expression is a suitable approach for blocking T-DNA transfer, and would ultimately attenuate the tumorigenesis efficiency of A. tumefaciens.
It has been evidenced that the vir gene expression are encoded by the conjugative tumor-inducing (Ti) plasmid, and the dissemination of which was mediated by the quorum sensing (QS) signal 3-oxo-octanoyl-L-homoserine lactone (3-oxo-C8-HSL) . The QS signal 3-oxo-C8-HSL is synthesized by the TraI protein, a LuxI homologue encoded in Ti plasmid. 3-oxo-C8-HSL binds to the receptor TraR and the formed complex binds to the promoter elements tra boxes, eventually initiates T-DNA transfer and motivates virulencerelated gene expression (Swiderska et al., 2001). Therefore, disrupting QS could be an attractive approach for controlling crown gall disease caused by A. tumefaciens.
Two basic approaches were employed for disrupting QS of A. tumefaciens, i.e., enzyme degradation and small molecules competitive binding (Uroz et al., 2009). The latter has been widely studied by applying chemically synthesized analog of the QS signals to bind to the receptors (Galloway et al., 2011). Though these synthetic analog have potential for acting as QS inhibitors, production costs and success rates make drugs from natural products more suitable (Kong et al., 2009). Endophytes are ubiquitous in all plant species and have proven to be potential sources of novel natural products and anti-QS agents for exploitation in medicine and agriculture (Yu et al., 2010). For example, a series of novel polyhydroxyanthraquinones with ant-QS potential were isolated from the metabolites of endophytic fungus Penicillium restrictum (Figueroa et al., 2014). The endophytic fungus Alternaria alternata produced bis (2-methylpropyl) ester, 2propyl tridecyl ester and 1,2-benzenedicarboxylic acid, which possessed anti-virulence and anti-biofilm capacities (Rashmi et al., 2018). Recently, 4-hydroxycinnamic acid (HA) was firstly isolated from the secondary metabolites of Phomopsis liquidambari S47, one endophytic fungus obtained from the leaves of Punica granatum. The structure of HA was elucidated by ESI-MS and NMR. HA has anti-QS potential against A. tumefaciens (Bodini et al., 2009). However, the underlying anti-QS mechanism and metabolic response of HA to A. tumefaciens have not been pinpointed. In this study, the anti-QS potential of HA was assessed by employing the AHL-responsive reporter strain A. tumefaciens A136 (pCF218/PCF372) (traI: lacZ) (McLean et al., 1997) and the possible metabolic mechanism of HA was investigated through 1 H NMR spectroscopy. No. 2 medium (Zhou et al., 2017), the metabolites were extracted with ethyl acetate and then purified by normal and reversed phase silica gel chromatography, HPLC, and Sephadex LH-20 gel permeation chromatography with A. tumefaciens KYC55 (pJZ372, pJZ 384, pJZ410) as the activity tracing strain (Zhou et al., 2017). The purified metabolites were identified by ESI-MS and NMR.

Growth Measurement
HA used in this study was purchased from Shanghai Yuanye Bio-Technology Co., Ltd. This compound is white needle crystal with purity > 98%. The minimum inhibitory concentration (MIC) of HA was determined using the broth microdilution method with an inoculum of 1-5 × 10 5 CFU/mL (Molitor, 2019). The growth analysis was determined by inoculating 0.1% overnight cultures of A. tumefaciens into AT medium (Kim et al., 2005) supplemented with different concentrations of HA. After incubation at 28 • C for 24 h, growth was determined by reading OD 620 . Each treatment was performed in triplicate.

β-Galactosidase Activity
Overnight cultures of A. tumefaciens A136 (100 µL) were inoculated into 100 mL of AT medium with or without HA. The signal molecule 3-oxo-C8-HSL (3 µM) was added into the culture and then cultivated at 28 • C for 17 h. Salicylic acid (SA, 0.10 mM) was used as the positive control. Each treatment was performed in triplicate. After cultivation, growth was evaluated by reading OD 620 (A 620 ). For β-galactosidase activity, 200 µL of cultures were added with 800 µL of Z-buffer, 100 µL of 0.1% SDS, 150 µL of chloroform, and 100 µL of ONPG (4 mg/mL). The mixture was incubated at 28 • C for several minutes (T), followed by the addition of 1 M sodium carbonate solution. After centrifugation, the supernatant was determined by reading OD 420 (A 420 ).The βgalactosidase activity was evaluated using the following formula (Stachel et al., 1985). (1)

Biofilm Formation
Biofilm formation was assayed in 96-well plate as described by Merritt et al. (2005). Briefly, A. tumefaciens C58 was cultivated in AT medium with 0.30, 0.60, and 0.80 mM of HA at 28 • C for 24 h. DMSO served as the negative control. After cultivation, the bacteria solution was removed and the planktonic bacteria were washed with PBS gently. Biofilms were dried at 60 • C, fixed with 200 µL of methanol, stained with 100 µL of 0.05% crystal violet, washed with PBS to remove excess dye, and quantified after solubilization of the dye with ethanol by reading the microplates at 570 nm. For microscopic analysis, the biofilms established in 24-well chambered cover slides were washed with PBS, fixed with 2.5% glutaraldehyde, dehydrated with graded ethanol, and subsequently freeze-dried (Zhou et al., 2018b). Biofilms were then gold-coated, and subjected to scanning electron microscopy (SEM, JSM6360, JEOL, Tokyo, Japan).

Acylated Homoserine Lactones Analysis
The anti-QS activity of HA was further confirmed by quantifying the 3-oxo-C8-HSL level secreted by A. tumefaciens C58 (wild strain) (Wood et al., 2001). Briefly, 0.1% overnight cultures of A. tumefaciens C58 was inoculated into AT medium with or without HA and then cultivated at 28 • C for 17 h. Each treatment was performed in triplicate. After centrifugation, the supernatant was extracted with an equal volume of acidified ethyl acetate. After evaporation, the residues were dissolved with methanol and 3-oxo-C8-HSL was quantified using liquid chromatography-tandem mass spectrometry (LC-MS/MS) (SHIMADZU) equipped with a C18-column (250 × 4.6 mm, 5 µm, Shimadzu, Tokyo, Japan) (Zhou et al., 2018b). Mobile phase A was formic acid (0.1%) and ammonium formate (5.0 mM) in water while mobile B was MeOH. The flow rate was 0.4 mL/min with 10 µL injection volume. The gradient was 1-5 min, 20-50% B; 5-20 min, 50-90% B. The eluates were determined with a ThermoFinnigan LCQ Classic system (San Jose, CA) using the positive mode. The nebulizer was 15 psi, drying gas 7 mL/min, and temperature 300 • C. Full-scan MS was from m/z 100 to 1,000. 3-oxo-C8-HSL was identified according to the fragment MS ions and the retention time of commercial standard (Sigma-Aldrich, United States). The area of the ion m/z 102 was selected to quantify 3-oxo-C8-HSL due to its specificity and better signalto-noise ratio. The extracted ion chromatograms were employed for area calculation.

Swimming and Chemotaxis
For swimming motility assay, 2 µL overnight cultures of A. tumefaciens C58 (OD 620 = 0.5) were dropped to the ATGN medium with different concentrations of HA as described by Merritt et al. (2007). Salicylic acid (0.10 mM) and DMSO served as the positive and negative control, respectively. The swimming diameter was determined after 24-h cultivation at 28 • C. Each treatment was performed in triplicate. Chemotaxis assay was performed by inoculating 2 µL overnight cultures of A. tumefaciens C58 on one side of the ATGN medium (without glucose) with different concentrations of HA as described by Ding and Christie (2003). One strip filter paper dipped with glucose was set as 4 cm aside from the inoculated bacterial cultures. After 24-h incubation at 28 • C, the chemotaxis diameter was measured. Each treatment was performed in triplicate.

Flagella Formation
After 17-h cultivation with or without HA, 50 µL of 37% methanol was added to 1 mL of bacterial cultures. The mixture was centrifuged at 5,000 rpm for 2 min and washed with distilled water. The collected cells were resuspended with distilled water and then dropped on a slide. After 5-min staining with the flagella staining solution (Beijing Solarbio Science & Technology Co., Ltd., China), flagella were detected with a light microscope (Nikon 80i, Japan). Each treatment was performed in triplicate.

Metabolic Analysis
A. tumefaciens C58 was cultivated with 0.60 mM of HA at 28 • C for 17 h. After cultivation, cells were collected and washed three times with precooled PBS. Cell pellets were homogenized with 3.8 mL of methanol/H 2 O (1/0.9) and then added with 4 mL of chloroform. The mixtures were centrifuged and the FIGURE 4 | Effect of HA on swimming motility (A), chemotaxis (B), and flagella formation (C). Images of (a-f) represented DMSO, salicylic acid (SA, 0.10 mM), 0.30, 0.45, 0.60, and 0.80 mM of HA treatment, respectively. The images of swimming motility and chemotaxis were obtained by a common camera. The flagella formation was observed using a light microscope.
upper layer was polled. After removal of methanol, samples were lyophilized and then resolved in D 2 O phosphate buffer for 1 H NMR analysis. Data processing and metabolites assignment were performed as described previously (Zhou et al., 2019a). The fold change values of metabolites between groups were calculated by the ratios of integral non-overlapping areas of each metabolite. The Benjamini and Hochberg (BH) method was used to adjust the p-values for controlling the false discovery rate in multiple comparisons using scripts written in R language. The fold change values of metabolites and associated p-values adjusted by BH methods in different group comparisons were visualized in fold change plots. Each treatment was performed 11 times.

Reactive Oxygen Species and H 2 O 2 Measurement
For ROS determination, the harvested cells were mixed with 6carboxy-2 ,7 -dichlorodihydrofluorescein diacetate (DCFH-DA, 1 mM). After 30-min cultivation at 28 • C, cells were collected, washed with distilled water, and then resuspended with PBS (1 mL). ROS was quantified using a Hitachi 2,700 fluorescence spectrophotometer (Hitachi, Japan) as described previously (Zhou et al., 2019b). Each treatment was performed in triplicate.
H 2 O 2 was determined according to Gonzalez-Flecha and Demple with minor modifications (Gonzalez-Flecha and Demple, 1997). Briefly, bacterial cultures were pelleted and resuspended with PBS. Intracellular H 2 O 2 passed through the membranes and equilibrated with the buffer. After 1-min centrifugation at 6,000 g, the suspensions were used for H 2 O 2 analysis employing the horseradish peroxidase-scopoletin method. Each treatment was performed in triplicate.

Quantitative Real-Time PCR
After cultivation, cells were harvested and total RNA was extracted using RNA extraction kit (Tiangen Biotech, Beijing, China). The Quantitative Real-Time PCR (qRT-PCR) assay was performed as described by Zhou et al. (2018b) with lepA (atu0241) set as the reference gene . The primers were listed in Supplementary Table 1. Each treatment was performed in triplicate.

Docking Analysis
Discovery Studio 4.0 program was used for docking simulation. The structures of HA and 3-oxo-C8-HSL were performed by ChemBioDraw 12.0. The target TraR (NDB code PD0654) was docked with 3-oxo-C8-HSL and HA, respectively, and the scores we recorded as kcal/mol (Zhou et al., 2018a).

Tobacco Infection Assay
Overnight cultures of A. tumefaciens C58 were inoculated into AT medium with 0.30 and 0.60 mM of HA. After cultivation at 28 • C and 180 rpm for 17 h, cells were collected, washed with sterile distilled water, and then resuspended with PBS to obtain an OD 620 = 0.1. Stems of 4-week-old tobaccos were inoculated with bacterial suspensions using a needle as described by Dandurishvili et al. (2011) and Liu et al. (2012). The black control was inoculated with sterile PBS. Photograph and tumor weight were recorded after inoculation for 4 weeks. Each treatment was performed in triplicate.

Purification and Identification of Active Metabolites
A total of 61 compounds were purified from the metabolites of P. liquidambari by repeated silica gel, RP-18 gel, Sephadex LH-20, and HPLC with A. tumefaciens KYC55 as the reporter strain. Limited by their content and spectroscopic techniques, 10 compounds were elucidated by ESI-MS and NMR spectra. Among these 10 compounds, 4-hydroxycinnamic acid (HA) showed the strongest anti-QS capacity against A. tumefaciens. The spectroscopic data of HA were presented as follows.

Minimum Inhibitory Concentration Determination
The MIC of HA against A. tumefaciens was > 1.20 mM. The growth curve was obtained using HA at sub-MIC concentrations.
Results indicated that exposure to HA at 0.30, 0.45, and 0.60 mM had no inhibitory impact on cell growth (Figure 1). However, treatment with HA at 0.80 mM showed inhibitory effect on cell growth (Figure 1).

β-Galactosidase Activity
The ani-QS activity of HA was determined through βgalactosidase activity using A. tumefaciens A136 (traI::lacZ) as the AHL-responsive reporter strain. Results indicated that treatment with HA at 0.30, 0.45, 0.60, and 0.80 mM reduced β-galactosidase activity by about 54, 62, 74, and 88%, respectively (Figure 2A). The anti-QS activity of HA was more effective than that of salicylic acid, whose exposure resulted in 48% inhibition of βgalactosidase activity.

Biofilm Formation
The inhibitory effect of HA on A. tumefaciens biofilm formation was presented in Figures 2B,C. Treatment with HA at concentrations of 0.30, 0.60, and 0.80 mM significantly reduced the formation of biofilms by 40, 61, and 83%, respectively ( Figure 2B). In addition to quantitative analysis, treated biofilms were also visualized using SEM. The SEM images showed thick biofilms in the control experiment (Figure 2Ca), whereas HA exposure at 0.30, 0.60, and 0.80 mM Values with asterisk symbols denoted extent of significance: *p < 0.05, **p < 0.01, and ***p < 0.001.
significantly removed the microbes attached to the glass surface (Figures 2Cb-d).

Acylated Homoserine Lactones Production
The anti-QS activity was further confirmed by determining AHL level produced by A. tumefaciens. Results indicated that the signal molecule 3-o-C8-HSL was secreted in the supernatant of A. tumefaciens C58 (Figures 3A,B). Treatment with HA at 0.30, 0.45, and 0.60 mM remarkably reduced the peaks and areas of 3-oxo-C8-HSL ( Figure 3A). Relative quantification by HPLC chromatograms indicated that HA treatment resulted in the reduced production of 3-oxo-C8-HSL by about 50, 60, and 80%, respectively ( Figure 3C).

Motilities and Flagella Formation
Swimming, chemotaxis, and flagella are essential for A. tumefaciens to survive for nutrients and infect host (Erhardt, 2016). Therefore, interfering with swimming, chemotaxis, and flagella formation may suppress the infection ability and reduce the pathogenicity of A. tumefaciens. Results indicated that exposure to HA at 0.30, 0.45, 0.60, and 0.80 mM, the swimming motility ( Figure 4A), chemotaxis (Figure 4B), and flagella formation (Figure 4C) of A. tumefaciens were remarkably suppressed. The suppressed ability of HA was superior to that of salicylic acid.

Metabolic Profile
1 H NMR-based metabolomics approach was employed to evaluate the metabolic changes of A. tumefaciens C58 exposure to HA (Figure 5). A remarkable decrease in cholate, putrescine, sarcosine, N, N-dimethylglycine, ethanolamine, choline, glycine, and formate, and a significant increase in glutamate, glutathione, dimethylamine, methanol, UDP-galactose, adenosine, AMP, and xanthine were observed after HA treatment (Table 1 and Figures 6B-D). The PCA score plot presented a significant separation between HA and the DMSO-treated groups ( Figure 6A). This indicated that the metabolism of A. tumefaciens C58 was deeply disturbed after HA treatment. The detailed assignment of metabolites and their fold changes were displayed in Table 1.

Reactive Oxygen Species and H 2 O 2 Analysis
The production of ROS and H 2 O 2 with HA treatment was displayed in Figure 7A. HA exposure at 0.60 mM remarkably enhanced the production of ROS and H 2 O 2 . The result implied that A. tumefaciens C58 underwent severe oxidative stress after exposure to HA.

Gene Expressions
qRT-PCR was used to evaluate the impact of HA (0.60 mM) on the transcriptional levels of nine genes traR, traI, virA,   (Figure 7B). The obtained data indicated that HA treatment resulted in the breakdown of QS, lead to enhanced oxidative stress, and would inevitably reduce the pathogenicity of A. tumefaciens C58.

Docking Simulation
Docking simulation was employed for assessing the proposed binding domain and binding ability of HA to TraR. The amino group of 3-oxo-C8-HSL showed H-binding interaction with Asp70 at a distance of 2.81 Å and the carbonyl group exhibited H-binding interaction with Thr129 at a distance of 3.26 Å (Figure 8). The two hydroxyl groups of HA displayed H-interactions with Asp70 and Gln58, respectively (Figure 8). The binding energy between 3-oxo-C8-HSL and TraR was −8.09 kcal/mol, whereas HA was −6.57 kcal/mol. The result indicated that 3-oxo-C8-HSL has stronger binding affinity to TraR than HA.

Pathogenicity Suppression Assay in Tobacco
The inhibitory effect of HA on pathogenicity was evaluated by inoculating A. tumefaciens C58 into tobacco stems. Results indicated that the size and weight of crown galls from infection with HA-treated A. tumefaciens C58 were evidently smaller compared with the untreated control (Figure 9). The tumorigenic ability of the HA-treated strain was obviously attenuated. The results indicated that HA had the potential to function as a pesticide in treating A. tumefaciens infection.

DISCUSSION
A. tumefaciens is a soil-borne phytopathogen that infects a wide range of dicotyledonous plants by transferring and integrating its own T-DNA into plant genome (Gohlke and Deeken, 2014). The expressions of the integrated genes intensified the synthesis of auxin and cytokinin, and then induced the proliferation of crown galls, activated the expressions of virulence-related genes, and ultimately caused the reduction of crop yield. It has been evidenced that T-DNA transfer and virulence activation were well correlated with QS (Fuqua and Winans, 1994). Thus, suppressing QS would be a compelling alternative for controlling crown gall disease caused by A. tumefaciens. In this study, a QS inhibitor named 4-hydroxycinnamic acid (HA) was firstly isolated from the metabolites of P. liquidambari S47 and its anti-QS, anti-virulence, and metabolic mechanisms were determined. The potential anti-QS activity of HA was firstly evaluated by β-galactosidase assay. The reduced β-galactosidase activity indicated that HA might disrupt the QS of A. tumefaciens. As the significance of AHL in coordinating conjugal transfer of the tumor-inducing plasmid and mediating the expressions of a battery of genes involved in virulence (Swiderska et al., 2001), the effect of HA on AHL secretion was measured. The notable suppression of AHL secretion further indicated the potent anti-QS activity of HA. The result was consistent with the reduced transcriptional levels of traI and traR. However, docking analysis indicated that the binding affinity of HA to TraR was weaker than that of 3-oxo-C8-HSL. Therefore, we speculated that the anti-QS potential of HA was obtained by inhibiting traI and traR expressions rather than competitive binding.
In addition, biofilms, flagella and motilities are essential for drug resistance, sensing plant-derived signals, striving for nutrients, and infection process (Ding and Christie, 2003). Our result showed that HA treatment resulted in a significant inhibition in biofilm formation, swimming, chemotaxis, and flagella formation. The inhibition of biofilm formation would The impact of HA on metabolic profile of A. tumefaciens was deeply evaluated by 1 H NMR-based analysis. Results presented that the metabolic system of A. tumefaciens was significantly disturbed with HA exposure. Some of the altered metabolites were related to quorum sensing, protein synthesis, and oxidative stress, including putrescine, valine, ethanolamine, and choline. It has been evidenced that the synthesis of putrescine was deeply affected by QS signals (Zhu et al., 2016). The reduced putrescine was consistent with the inhibited β-galactosidase activity and reduced AHL secretion. Valine is one of the branched-chain amino acids and acts as vital roles in maintaining normal functions of cells (Zhou et al., 2019a). The changed valine might indicate the changed cell function caused by HA. Ethanolamine is an essential component of membrane and act as an important role in maintaining membrane integrity (Zhou et al., 2019a). The notable reduction in ethanolamine indicated the intensified oxidative stress caused by HA. To defend against oxidative damage and repair the damaged membrane, the consumption of ethanolamine was enhanced. This speculation was validated by the increased ROS and H 2 O 2 levels. In addition, the significant reduction of choline, N, N-dimethylglycine, and sarcosine, and the increase of dimethylamine, glutamate, and glutathione also confirmed the intensified oxidative damage.
It is known that excessive oxidative damage can lead to cell death. However, in this study, HA treatment at 0.60 mM had no inhibitory impact on cell growth, which was evidenced by the growth profile, plate counting assay (Supplementary Figure 2A) and ATP investigation test (Supplementary Figure 2D). Figure 2B)    cell membrane. Since HA is not bactericidal at 0.60 mM and the fluorescence intensity in HA-treated group was just 18% higher than that of DMSO-treated group, we can draw a conclusion that HA treatment destroyed the integrity of cell membrane. In addition, ROS was determined after treatment with the lethal concentration of 1.20 mM (data not shown). HA exposure at 1.20 mM resulted in the ROS production of 501 ± 37 fluorescence intensity, which was significantly higher than that of 0.60 mM of HA treatment (106 ± 11 fluorescence intensity). Thus, we speculated that the bactericidal effect of HA on A. tumefaciens is due to the excessive oxidative damage, while HA at sublethal concentration brought no excessive oxidative damage. In other words, the oxidative damage caused by HA at 0.60 mM was not enough to kill cells, but only changed the membrane composition and enhanced cell membrane permeability without sterilization. This speculation was in accordance with the study of Zhou et al. (2018b), who reported that the quorum sensing inhibitor hordenine could notably enhance the susceptibility of the preformed biofilms to ciprofloxacin by changing the permeability of membranes, as evidenced by the reduced level of ethanolamine. In addition, Cheng et al. (2020) also reported that the quorum sensing inhibitor dimethylaminocinnamic acid could destroy the integrity of cell membrane by reducing ethanolamine level with no sterilization.

The orange cells (Supplementary
Choline is the component of phospholipids and is responsible for keeping integrity and permeability of membrane (Zhou et al., 2019a). The reduced choline level indicated that the oxidative stress caused by HA destroyed the structure of membrane. To maintain oxidative balance and repair the damaged membrane, choline consumption was enhanced. The decomposition of choline resulted in the enhanced level of dimethylamine as dimethylamine was the downstream metabolite of choline (Dumas et al., 2006). The enhanced oxidative stress was further evaluated by determining the levels of glutamate and glutathione, and the transcriptional level of gene sodB. It has been proven that the activity of superoxide dismutase (SOD) was mediated by QS (Hassett et al., 1999). In this study, the transcriptional level of sodB encoding SOD was significantly repressed and this result was in line with the dysfunctional QS. The enhanced glutamate and glutathione and the suppressed sodB would inevitably intensified oxidative stress and ultimately lead to the damage of cell membrane, disorder of metabolic profile, and reduced pathogenicity of A. tumefaciens.
In addition, it has been evidenced that 3-oxo-C8-HSL synthesis is affected by fatty acid metabolism (Dong et al., 2017). The intermediate products of fatty acid metabolism can provide the matrix for 3-oxo-C8-HSL synthesis. The suppressed secretion of 3-oxo-C8-HSL indicated that fatty acid metabolism might be inhibited. The inhibition of fatty acid metabolism would change cell membrane composition and this was verified by the reduced ethanolamine and choline.
As a powerful antioxidant in organism, betaine acts as an important role in maintaining cell integrity and normal function . Betaine can be transformed into N, N-dimethylglycine with the catalysis of betaine homocysteine methyltransferase, and N, N-dimethylglycine was then transformed into sarcosine and glycine. The decrease of betaine can be attributed to choline reduction as betaine is the oxidative product of choline in organisms. To defend against oxidative stress and repair the damaged cells caused by free radicals, betaine was excessively consumed.
HA treatment also caused disorder of energy metabolism, which was evidenced by the increased succinate. Succinate is an essential intermediate involved in tricarboxylic acid (TCA) cycle. The altered succinate would inevitably block TCA cycle. As the most important pathway for producing energy, the block of TCA cycle would inevitably affect energy metabolism, lead to energy deficit, result in dysfunction of cells, and ultimately reduce virulence and pathogenicity of A. tumefaciens. This hypothesis was confirmed by the attenuated crown gall disease. The blocked TCA cycle and insufficient energy supply lead to intensified anaerobic respiration for compensation, which was supported by the increased level of ethanol.
Adenosine, AMP and xanthine are intermediate metabolites of purine nucleotide. AMP can be converted to adenosine by dephosphorylation, and then transformed into inosine by deaminase. Inosine is subsequently transformed into xanthine through hydrolysis and oxidation (Zhou et al., 2019a). As is known that AMP is derived from cyclic AMP (cAMP) through cAMP-specific phosphodiesterase and cyclic 3 ,5 -phosphodiesterase. It has been evidenced that cAMP contributes to QS, biofilm formation, motility, and expressions of genes responsible for flagella, toxins, and energy supply (Liang et al., 2007). The increase of inosine, AMP, and xanthine indicated that the catabolism of cAMP was intensified. The excessive catabolism of cAMP would lead to dysfunctional QS and reduced pathogenicity of A. tumefaciens. This speculation was supported by the reduced AHL, suppressed virulence factors, and weakened infection in plant.
In sum, 4-hydroxycinnamic acid (HA) was firstly isolated from the metabolites of endophytic fungus P. liquidambari and its anti-QS, anti-virulence potential and metabolic mechanism were deeply investigated. A schematic diagram of the disordered metabolic mechanism was displayed in Figure 10. HA treatment resulted in the dysfunction of QS. The dysfunctional QS intensified oxidative stress, caused deficiency of energy supply, lead to disorder of protein metabolism and nuclear acid metabolism, and ultimately reduced virulence and crown gall disease caused by A. tumefaciens. Therefore, HA may act as an efficient pesticide for controlling A. tumefaciens infection.

DATA AVAILABILITY STATEMENT
The original contributions presented in the study are included in the article/Supplementary Material, further inquiries can be directed to the corresponding author/s.


Immune remodeling and atrial fibrillation

Atrial fibrillation (AF) is a highly prevalent arrhythmia that causes high morbidity and mortality. However, the underlying mechanism of AF has not been fully elucidated. Recent research has suggested that, during AF, the immune system changes considerably and interacts with the environment and cells involved in the initiation and maintenance of AF. This may provide a new direction for research and therapeutic strategies for AF. In this review, we elaborate the concept of immune remodeling based on available data in AF. Then, we highlight the complex relationships between immune remodeling and atrial electrical, structural and neural remodeling while also pointing out some research gaps in these field. Finally, we discuss several potential immunomodulatory treatments for AF. Although the heterogeneity of existing evidence makes it ambiguous to extrapolate immunomodulatory treatments for AF into the clinical practice, immune remodeling is still an evolving concept in AF pathophysiology and further studies within this field are likely to provide effective therapies for AF.

Introduction
Atrial fibrillation (AF) is the most common sustained arrhythmia in clinical practice and is associated with complications, such as heart failure and stroke (Lippi et al., 2021). In recent decades, the combination of "trigger" and "substrate" has been considered the major cause for the initiation and maintenance of AF. In atypical sites, including pulmonary vein ostia, coronary sinus, ligament of Marshall, abnormal automaticity or early and delayed afterdepolarizations induce ectopic activity and then initiate AF (Santangeli and Marchlinski, 2017). The substrate, which manifests as atrial remodeling, increases the likelihood of ectopic firing or re-entry. There are at least three main forms of remodeling: electrical remodeling, structural remodeling and autonomic neural remodeling. Electrical remodeling manifests as changes in the number and distribution of ion channels and gap junction proteins, leading to a shortened atrial effective refractory period (AERP) and increased AERP dispersion (Grunnet et al., 2012). Structural remodeling is characterized by progressive collagen deposition and atrial fibrosis, which is a consequence of cardiac fibroblast activation and extracellular matrix metabolic dysfunction (Sohns and Marrouche, 2020). Autonomic neural remodeling presents primarily as nonhomogeneous nerve sprouts and an imbalance in sympathetic and parasympathetic activity, which can be related to the release of nerve growth factor (NGF) (Shen et al., 2011). Interestingly, these mechanisms are not independent, and the development of AF arises from their mutual promotion and comprehensive effects (Wijesurendra and Casadei, 2019;Kusayama et al., 2021). Furthermore, recent studies have shown that the immune system changes considerably and plays an essential role in the pathophysiological process of AF. Here, we regard the change of the immune system in AF as another form of remodeling and known as immune remodeling. It refers to the recruitment and activation of immune cells induced by various factors as well as the alternation in immune molecular secretion, which shapes a new immune network during AF Xiao et al., 2021). Moreover, immune remodeling goes throughout the whole process of AF development and maintenance (Miyosawa et al., 2020). On the one hand, immune remodeling regulates the known electrical, structural and neural remodeling to participate in the development of AF; on the other hand, the AF-related pathological changes including fibrosis act as a positive regulator of immune remodeling and further promote the maintenance of AF (Wernli et al., 2009;Rao et al., 2013;Fu et al., 2015) (Figure 1). Importantly, unlike atrial remodeling, immune remodeling is not limited to the atria and its effects on the peripheral circulation can not only facilitate clinical diagnosis but also show that AF should be considered as a systemic disease. However, the cause-effect-cause complexity makes it a bit more difficult to strictly distinguish between those events that might be the result of AF-induced immune remodeling or immune remodeling induced AF. Therefore, related immunology studies will greatly improve understanding of AF.
In this review, we focus on the association between immune remodeling and AF. First, we begin with a fundamental introduction to cardiac immunology components; then, based on clinical research data, the evidence of immune remodeling during AF is described in detail. Furthermore, we link immune remodeling to atrial electrical, structural and neural remodeling at the cellular and molecular levels. Finally, we summarize some potential therapies and aim to provide more precise and effective targets for AF treatment.

Immune cells in cardiac homeostasis
Recent insights into the immune system and cardiology have suggested that immune cells are integral components for maintaining homeostasis in cardiac tissue (Hulsmans et al., 2017). The study on healthy adult mice showed that the immune cells constituted 4.7% ± 1.5% of the cardiac tissues. The frequencies of major immune cells in cardiac muscle were 12-fold higher relative to the skeletal muscles (Pinto et al., 2016;Ramos et al., 2017). Among them, approximately 81.4% ± 1.4% were myeloid cells, 8.9% ± 0.6% were B cells, 3.1% ± 0.4% were T cells and 6.6% ± 0.6% were non-myeloid/lymphoid immune cells (Pinto et al., 2016). However, this previous study is limited to ventricles without atria and valves. Another study on the adult human heart demonstrated the presence of 11 major cell types, including atrial cardiomyocytes, ventricular cardiomyocytes, fibroblasts, endothelial cells, and immune cells. Among them, myeloid and lymphoid immune cells accounted for 5.3% of the ventricular tissues and 10.4% of the atrial tissues (Litviňuková et al., 2020), which is in line with the findings in mice, which suggest that immune cells occupy a certain substantial proportion of the healthy heart. However, some differences may exist across species. For example, it was thought that the density of mast cells in the hearts of canines and humans was higher relative to the mouse heart (Gersch et al., 2002;Ingason et al., 2019). The distribution of immune cells in distinct cardiac areas is also non-homogeneous. Only a few studies have characterized the proportion of immune cell subsets in the atria. A precise understanding of immune cell subsets and their proportions in atria will pave the way for illustrating the mechanism underlying AF. In addition to the myocardium tissues, the pericardial fluid and adipose tissues contain some immune cells, which are important sources of tissue infiltration under stress (Butts et al., 2017;Horckmans et al., 2018). The origin, phenotypes, and functions of cardiac immune cells have been summarized in previous reviews (Lavine et al., 2018;Swirski and Nahrendorf, 2018;Varricchi et al., 2020).

FIGURE 1
The association between immune remodeling and AF. This figure recapitulates briefly the association between immune remodeling and AF. Immune remodeling regulates atrial electrical, structual and neural remodeling to promote AF. In turn, AFrelated pathologic changes including fibrosis can induce immune remodeling, which forms a positive circle loop. Abbreviations: AF, atrial fibrillation.

Immune remodeling during atrial fibrillation
Evidence for immune remodeling Immune remodeling is a multidimensional pathological process. The changes in the composition and number of immune cells can be regarded as morphologic or structural evidence for immune remodeling, while alternations in immune molecules can be considered as functional evidence of immune remodeling. In this review, we mainly summarize the current clinical evidence to support the concept of immune remodeling in AF. Table 1 provides a summary of studies on the change in immune cell types in AF. CD45 is common to all inflammatory cells. As early as 2008, it was found that, as compared to that in individuals with normal sinus rhythm (SR), the infiltration of CD45 + cells was increased markedly in the atria of AF patients. Among those with AF, no difference in CD45 + inflammatory cell infiltration was observed between the left and right atria (Chen et al., 2008). A subsequent study by Yamashita and colleagues also supported this conclusion. They further demonstrated that in AF specimens, the infiltration of CD45 + and CD68 + cells in the atria endo-and sub-endomyocardium was predominantly high relative to the mid-myocardium and proposed that immune cells were recruited across the atrial endocardium during AF (Yamashita et al., 2010). Moreover, the number of peripheral CD45 + cells was increased significantly in patients with AF (Aguiar et al., 2019). Dendritic and mast cells have been observed in the atria of AF patients. The number of dendritic cells in AF patients was higher relative to those with SR, whereas the number of mast cells was similar (Smorodinova et al., 2017). Relative to the subjects with SR, the number of CD3 + T cells was increased significantly in the atrial tissue of patients with AF, which has been confirmed in several clinical studies (Smorodinova et al., 2017;Hohmann et al., 2020;Wu et al., 2020). However, whether the number of CD3 + T cells differs significantly in the AF subgroups remains controversial. A previous study argued that the number of CD3 + T cells increased from patients with SR to paroxysmal AF (pAF) and persistent AF (peAF), respectively. The number was lower in patients with permanent AF (permAF) relative to those with peAF (Hohmann et al., 2020). However, another study suggested that no statistical difference existed in CD3 + T cell infiltration between pAF and peAF/permAF (Wu et al., 2020). Differences in these findings may be due to the clinical heterogeneity of samples and the specificity of the antibodies. After all, these findings are highly dependent on the immunohistochemical and flow cytometry methods. Additionally, CD20 + B cells are occasionally present as small clusters in the epicardial layer and are very rare in the myocardium (Hohmann et al., 2020). As described previously, adipose tissue usually contains a far greater number of immune cells. The number of neutrophilic granulocytes and lymphocytes was higher in the atrial fat tissue of AF patients relative to SR individuals (Begieneman et al., 2015). Several animal studies have demonstrated the change in proportions of cardiac immune cells (especially macrophages) during the onset and maintenance of AF (Sun et al., 2016;He et al., 2021). In addition to routine inflammatory cells, activated platelets have also been proposed to be associated with AF. Tolllike receptors (TLRs) are expressed on the surface of platelets and participate in the platelet activation and thrombosis (Dib et al., 2020). A study found that compared with control group, peripheral and left atrial platelet TLR2 and TLR4 levels were significantly higher in AF patients. The above indicators were higher in atrium of peAF than that in pAF (Gurses et al., 2018). Another study in 2020 showed that increased platelet activation was found in peripheral blood from patients with hypertensive AF and the platelets were largely accumulated in these atriums , which implied the potential relation between platelets and AF. Taken together, the composition and number of immune cells are altered during AF. Functionally, inflammation is a primary, nonspecific response to the activation of innate and/or adaptive immunity. Several studies on the inflammation status of AF have been summarized in previous literature (Patel et al., 2010;Hu et al., 2015;Sagris et al., 2021). In this review, we highlight some direct evidence of the pathological alterations of immune molecules in AF (Table 2). In innate immunity, clinical studies showed that patients with AF had higher levels of circulating blood C-X-C motif ligand 1 (CXCL-1) and CXCL-12, which are critical regulators of monocyte/macrophage mobilization (Li et al., 2016;Zhang et al., 2020a). Galectin-3 is a β-galactoside binding lectin secreted by macrophages and its elevated levels can predict the progression from pAF to peAF . Macrophage migration inhibitory factor (MIF), a chemokine-like inflammatory cytokine, was also highly expressed in patients with AF, and AF progression corresponds to augmented MIF concentrations (Wan and Li, 2018). Additionally, as compared to the individuals without AF, atrial neutrophil extracellular traps and elevated serum myeloperoxidase (MPO) levels were frequent in patients with AF, which suggested infiltration and activation of neutrophils (Rudolph et al., 2010;Holzwirth et al., 2020). In adaptive immunity, it has previously been shown that patients with AF have higher levels of autoantibodies, including anti-β1adrenergic receptor (anti-β1-AR) and anti-M2-muscarinic receptor (anti-M2-R) . Anti-M2-R can predict the degree of left atrial fibrosis in pAF patients and β1-AR autoantibody may promote the development of AF by regulating atrial fibrosis Shang et al., 2020). Moreover, the levels of circulating immunoglobulin-free light chains, kappa and lambda, were higher in individuals with AF (Matsumori et al., 2020). These alternations are inconsistent with the findings for rare B cells in the atria. This phenomenon can be explained by the fact that circulating antibodies but not local B cells are associated with AF development. A matched casecontrol study assessed the association of Th17-related cytokines [including interleukin-17A (IL-17A), IL-17F, IL-21, and IL-22] with AF and observed elevated plasma levels of Th17-related cytokines were independently related to high risk of AF (Wu et al., 2016). These molecules participate in the atrial electrical and structural remodeling and facilitate the development of AF substrates. Overall, the immune microenvironment, including immune cells and molecules, change both locally and systemically during AF.

Mechanism of immune remodeling
The precise mechanisms of immune remodeling during AF are still elusive. On the one hand, it is possible that some risk factors and inflammation-related conditions, including hypertension, coronary atherosclerosis, obesity, sepsis and obstructive sleep apnea, can increase the release of damageassociated molecular patterns and/or pathogen-associated molecular patterns, leading to the activation of immune responses (Sirisinha, 2011). Cardiac surgical stimulation is also a potential trigger for immune cell activation, which explains the increased circulating immune cells and postoperative AF incidence after coronary artery bypass grafting (Hammer et al., 2021). Additionally, atrial rapid and irregular electrical activity causes intracellular calcium overload, oxidative stress and cell apoptosis, which in turn lead to more resident immune cell activation and cytokine release (Van Wagoner, 2008;Hu et al., 2015). This allows the formation of positive feedback loops between immune activation and AF. On the other hand, the function of negative regulators is impaired during AF, as shown by a Abbreviations: AF, atrial fibrillation; anti-β1-R, anti-β1-adrenergic receptor; anti-M2-R, anti-M2-muscarinic receptor; CXCL, C-X-C motif ligand; CXCR, C-X-C motif receptor; ELISA, enzyme-linked immuno sorbent assay; FLC, flow cytometry; IF, immunofluorescence; LAA, left atrial appendage; MIF, macrophage migration inhibitory factor; MPO, myeloperoxidase; pAF, paroxysmal AF; peAF, persistent AF; permAF, permanent AF; SDF-1α, stromal cell-derived factor-1α.
Frontiers in Physiology frontiersin.org 04 lower number of Treg cells and limited function of antiinflammatory mediators, including IL-4 and IL-10 (Sulzgruber et al., 2017). This point has been well proven by the finding that the depletion of spleen-derived IL-10 can augment AF vulnerability . In addition, the programmed death-1(PD-1) and its ligand PD-L1 have recently been highlighted as critical regulators that maintain this immune balance by negatively regulating T cell activation, proliferation and cytokine production. The downregulation of the PD-1/PD-L1 signaling pathway in AF partially participates in AF pathogenesis . Moreover, the imbalance in autonomic nerve system (ANS) is associated with the development and maintenance of AF (Kusayama et al., 2021), which may be an important cause of immune remodeling.

Immune remodeling and electrical remodeling
Recent investigations have revealed that the immune system has an impact on atrial electrophysiology (Figure 2). With the activation of immune cells, a large amount of pro-
Frontiers in Physiology frontiersin.org inflammatory factors, including tumor necrosis factor-α (TNF-α), MIF, IL-1β, IL-6 and galectin-3 are released subsequently during AF, which can induce atrial electrical remodeling. Ca2 + -associated abnormalities play a vital role in delayed afterdepolarizations and triggered activities (Schober et al., 2012). TNF-α can disrupt the intracellular calcium homeostasis in atrial myocytes by repressing the expression of T-type calcium channel α1G subunit (TCCA1G) and sarcoplasmic reticulum Ca-ATPases (SERCA2a) (Kao et al., 2010;Rao et al., 2016). TNF-α administration to the pulmonary vein cardiomyocytes affects multiple ionic currents (reduced I Ca-L and increased I to ), induces delayed afterdepolarizations, thereby enhancing arrhythmogenic activity (Lee et al., 2007). MIF treatment of HL-1 atrial myocytes increases calcium transients and sarcoplasmic reticulum calcium levels by inducing the expression of ryanodine receptor 2 (RyR2) (Cheng et al., 2020). In addition, activated immune cells such as macrophages prime the assembly of the NLRP3 inflammasome via TLR4 or nuclear factor-κB signaling, and trigger the release of IL-1β and IL-18. Macrophage-derived IL-1β can hinder quaking protein binding to the α1C subunit of L-type calcium channel (CACNA1C) and decrease calcium channel expression (Sun et al., 2016). Although macrophage NLRP3 activation is insufficient to cause AF, it has been shown that M1 macrophage-derived exosomes mediate cardiomyocyte NLRP3 activation by transferring miR-29a, while cardiomyocyte NLRP3 activation can upregulate the expression of RyR2 and promote abnormal sarcoplasmic reticulum Ca2 + release (Yao et al., 2018;. Moreover, galectin-3 is also an important contributor for atrial electrical remodeling. Galectin-3treated HL-1 myocytes have a shorter action potential duration, smaller I Ca-L current, increased sarcoplasmic reticulum calcium content and ultrarapid delayed rectifier potassium current than control cells have. Specific neutralization of its membrane surface receptor CD98 significantly weakens galectin-3-induced Ca 2+ handling imbalance (Cheng et al., 2022). Consistent with these results, Galectin-3 inhibitor GMCT treatment could mitigate pacing-induced electrical remodeling and abnormal Ca 2+ handling in a sheep model (Takemoto et al., 2016). The altered expression and distribution of connexins (Cx) on atrial myocytes disable gap junctional intercellular communication and reduce conduction velocity in the atrium, leading to increased vulnerability to AF (Nagibin et al., 2016). Mouse recombinant MIF can concentration-dependently downregulate Cx43 expression in atrial myocytes by activating ERK1/2 kinase . A recent study verified that elevated IL-6 levels rapidly lowered the expression of cardiac Cx43 and Cx40 (Lazzerini et al., 2019). In addition, TNF-α has the ability to change the expression and distribution of Cx43 and Cx40 (Liew et al., 2013). In an Ang II-infused hypertensive mouse model, Cx43 delocalization was obvious, while adoptive transfer of Treg cells induced normal Cx43 localization at the intercalated disk regions (Kvakan et al., 2009), which indicated that a reduced proportion of Treg cells in patients with AF might promote electrical remodeling by controlling Cx43 (Sulzgruber et al., 2017). Overall, immune cell-mediated electrophysiology and inflammatory response promote atrial electrical remodeling in an indirect or direct manner. Moreover, tachypacing of HL-1 atrial myocytes or Ang II-treated atrial myocyte-derived exosomes can promote M1 macrophage polarization (Sun et al., 2016;Cao et al., 2021). There is a reciprocal interaction between immune remodeling and electrical remodeling. There is no doubt that deep and systematic research on immuneelectrophysiology will provide new perspectives for AF treatments.

Immune remodeling and structural remodeling
Numerous studies have indicated that the infiltration of immune cells participates in atrial fibrosis and this effect depends mainly on the secretion of the cytokines (Figure 3). Macrophages, as major sources of transforming growth factor-β1 (TGF-β1) during the fibrotic process, can induce fibroblast-tomyofibroblast differentiation (Fadok et al., 1998). TNF-α is also involved in the pathogenesis of atrial fibrosis through activation of the TGF-β signaling pathway and increased secretion of matrix metalloproteinases (MMPs) (Liew et al., 2013). Galectin-3 produced by macrophages interacts with TGF-β and induces atrial fibrosis by stimulating the downstream TGF-β1/Smad pathway (Xiao et al., 2020). In addition, various chemokine receptors, including C-X-C chemokine receptor 2 (CXCR2), CXCR4, and CXCR6, are expressed on monocytes/ macrophages and involved in atrial fibrosis by mobilizing macrophages (Zhang et al., 2020a;Liu et al., 2021). It has been demonstrated that Ang II can induce chemokine expression in atrial fibroblasts, thereby inducing the chemotaxis of macrophages . There seems to be a positive feedback loop between macrophages and fibrosis, which is pivotal in the development of AF substrate.
Mast cells are involved in atrial structural remodeling. In a mouse model of diabetes, hyperglycemia led to mast cell infiltration in the atria, atrial fibrosis and increased AF susceptibility. Transgenic mast-cell deficiency reversed these pathological changes (Uemura et al., 2016). In a pressure-overload mouse model, activated mast cells promoted fibroblast activation and fibrosis by releasing platelet-derived growth factor α (PDGFα). Neutralizing the PDGFα receptor with a specific antibody alleviated atrial fibrosis and AF susceptibility (Liao et al., 2010). Activated Frontiers in Physiology frontiersin.org mast cells can also degranulate to produce some preformed mediators, including histamine, tryptase and chymase, which can mediate the progression of atrial fibrosis (Patella et al., 1995). Mast cells also serve as sources of TNF and IL-1β, promote fibrotic remodeling by inducing inflammation and MMP9 production (Mukai et al., 2018). However, due to some anti-fibrotic mediators in mast cells, the role of mast cells in fibrosis is controversial. Neutrophils release cytokines such as IL-6, TNF-α and MPO to accelerate atrial fibrosis. In Ang II-treated mice, neutrophil infiltration was accompanied by profoundly enhanced atrial fibrosis and elevated susceptibility to AF. MPO knockout blunted atrial fibrosis and protected Ang II-treated mice from AF by modulating MMP activity and hypochlorous acid formation (Rudolph et al., 2010). In addition, another study showed that the integrin CD11b/ CD18 mediate neutrophil infiltration and localization within the atrial tissue (Friedrichs et al., 2014), which provides a potential novel avenue of treatment in AF. Platelets are activated in patients with AF. Once activated, platelets can release a large number of pro-fibrotic cytokines and grow factors (including TGF-β1 and PDGF) into blood and local tissue (Mussano et al., 2016;Karolczak and Watala, 2021). In Ang II-infused mice model, both clopidogrel treatment and platelet-specific deletion of TGF-β1 reduced Ang II-induced atrial fibrosis and AF induction .
Multiple studies have also shown that T cell infiltration plays a functional role in cardiac fibrosis. This role is highly dependent on cardiac milieus and T cell subsets. In a myocardial infarction model, Th1 cells appeared to exert an anti-fibrotic effect by secreting interferon-γ. Conversely, FIGURE 3 Interactions of immune remodeling and structural remodeling. This figure summarizes the interactions between immune cells and CF during AF. Macrophage, mast cell, neutrophil and T cell can release pro-fibrotic factors to activate CF, leading to ECM deposition and atrial structural remodeling. Meanwhile, activated CF can promote the macrophage recruitment by releasing chemokines. Abbreviations: CCL, C-C motif ligand; CF, cardiac fibroblast; CXCL, C-X-C motif ligand; ECM, extracellular matrix; IFN-γ, interferon-γ; IL-4, interleukin-4; MPO, myeloperoxidase; PDGF, platelet-derived growth factor; TGF-β, transforming growth factor-β; TNF-α, tumor necrosis factor-α.

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Th2 cells counteracted to the Th1 response by secreting several pro-fibrotic cytokines (IL-4 and IL-13) (Bradshaw and DeLeon-Pennell, 2020). Lu et al. (2020) demonstrated that increased Th17 cells and decreased Treg cells aggravated myocardial fibrosis by activating the IL-17/ERK1/2-AP-1 pathway. This trend was also observed in immune remodeling in AF (He et al., 2018). However, the biological role of these subtypes has not been established in AF.

Immune remodeling and neural remodeling
The cardiac ANS plays a significant role in the occurrence and maintenance of AF. Recent studies have suggested that the immune system and ANS are intimately linked. Next, we will conclude the interactions between the immune system and ANS, and propose a number of blind spots in the existing experimental research on AF.
During immune remodeling, activated macrophages, mast cells and T cells can regulate neural remodeling by releasing inflammatory factors including NGF, IL-1β and IL-17A (Chaldakov et al., 2014;Lyu et al., 2020). An experimental study showed that catecholamine-induced inflammatory environment could promote the release of NGF from macrophages and regulate cardiac sympathetic remodeling (Lyu et al., 2020).The activation of NLRP3 inflammasome in macrophages can't only ignite inflammatory reaction but also facilitate the sympathetic innervation (Yin et al., 2017;Lee et al., 2021). The key role of macrophages in the regulation of neural remodeling has also been tested in vivo by clodronate liposomes-mediated macrophage depletion. It was revealed quite early that intravenous clodronate liposomes could decrease cardiac sympathetic axon density and NGF expression following myocardial infarction (Wernli et al., 2009). Local macrophage depletion in stellate ganglia could also restrain cardiac sympathetic sprouting and ventricular arrhythmias in heart failure . A recent study showed that clodronate liposome injection into the atria in canines significantly decreased AF vulnerability after acute stroke, but nerve density and NGF expression were not assessed . In addition, exogenetic IL-1β or IL-17A microinjection into the left stellate ganglion (LSG) promoted neuronal remodeling of the LSG and deteriorated ventricular electrophysiology by regulating the neural inflammation, which were improved by a specific neutralizing antibody Deng et al., 2019). The LSG also plays an important role in the initiation and maintenance of AF. However, atrial electrophysiology parameters were not measured at the same time. Although there is few direct evidence on immunemediated neural remodeling in AF, these findings indicate that it may be a novel vantage point for understanding AF mechanism.
The autonomic activity also plays a vital role in immune remodeling. Sympathetic nervous system activation can regulate immune system function through β-ARs, which exist in almost all immune cell types. β1-AR is primarily expressed in innate immune cells, where its activation can increase LPS-induced production of inflammatory mediators (Speidl et al., 2004). β2-AR is the most highly and widely expressed β-AR isoform in immune cells, and its effect is highly dependent on the initiation of downstream signaling (Yoshida et al., 2015;Grisanti et al., 2016). β3-AR has also been shown to be significant in mediating immune cell mobilization and egress from the bone marrow (Méndez-Ferrer et al., 2010). Additionally, some non-immune cells expressing β-AR also involve in ANS-mediated immune remodeling. Renal collecting duct epithelial cells express β2-AR and play a key role in the heart-brain-kidney network. When sympathetic nervous system activation stimulates the KLF5-S100A8-S100A9 pathway in collecting duct epithelial cells, renal macrophages produce TNF-α, which in turn stimulates renal endothelial cells to secrete colony stimulating factor 2 into the circulation, thereby activating cardiac resident macrophages (Fujiu et al., 2017). These results suggest that sympathetic neural activation can not only directly regulate cardiac immune cell activation but also indirectly modulates myelopoiesis and immune cell mobilization to the heart by affecting other organs, such as the bone marrow and kidney. SNS activation in AF is well known, along with migration and infiltration of immune cells and the inflammatory cascade. It was demonstrated that renal sympathetic denervation in canines could suppress AF and reduce the increasing trend of TNF-α and IL-6 induced by rapid atrial pacing (Wang et al., 2013). Acute middle cerebral artery occlusion in canines led to an increase in LSG activity, atrial β1-AR expression, atrial macrophage infiltration and AF vulnerability, while ablation of the LSG reversed these changes Yang et al., 2020). Although the definitive mechanism is not very clear, it is possible that the effect is related to sympathetic nerve-regulated immune remodeling. The parasympathetic nervous system regulates immune system function through nicotinic and muscarinic acetylcholine receptors (nAChRs and mAChRs) in most immune cells. The α7 subunit of nAChR (α7nAChR) is the most studied and involved in cholinergic anti-inflammatory pathway. Research of canines with rapid atrial pacing uncovered that low-level vagus nerve stimulation (LL-VNS) significantly suppressed atrial electrical remodeling and AF inducibility, accompanied by low levels of TNF-α and IL-6 in the left atria (Zhang S. J et al., 2021). Spinal cord stimulation facilitated the effect of VNS and reduced the induction of AF (Dai et al., 2017). Researchers also further demonstrated that median nerve stimulation could heighten cardiac vagal tone and atrial ACh levels, and reverse the enhanced inflammation response and AF inducibility by short-term rapid atrial pacing (Zhao et al., 2018). These results are associated with inflammatory macrophage inhibition mediated by the cholinergic anti-Frontiers in Physiology frontiersin.org inflammatory pathway. Despite suboptimal evidence, it could be speculated that the autonomic activity can regulate immune remodeling in AF.
The ANS activity is identified as a major component of the emotion response. Previous studies suggested that negative emotion including sadness, depression, anger and stress could increase the likelihood of symptomatic AF (Lampert et al., 2014). Depression is independently associated with AF recurrence after catheter ablation (Zhuo et al., 2020). The exact mechanisms are still mysterious. Recently, accumulating evidence have indicated the existence of immune remodeling during depression (Wittenberg et al., 2020). In major depressive disorder patients, anxious distress was highly associated with innate immune activation including higher levels of IL-6, TNF-α, monocyte chemoattractant protein-1 and increased number of activated monocytes in circulation (Gaspersz et al., 2017;Nowak et al., 2019). This shows that immune remodeling can be one of mediators of emotion-induced AF. In another study, major depressive disorder-derived monocytes displayed higher proportion of M1 polarization phenotype under standard culture conditions, but, higher M2 polarization when co-stimulated with autologous sera (Cosma et al., 2021). This fully reflects the plasticity of immune cells and potential value of immune regulation. However, the understanding for emotion-immune-AF correlation still be not thorough and deeper research is warrant.

Therapeutic potential of targeting immune remodeling
The mechanisms underlying AF have been studied in the last several decades. Although a wide range of modalities contributes to the management of AF, the treatment efficacy in patients with AF remains suboptimal. Immune remodeling induces inflammation and is highly associated with atrial electrical, structural, and neural remodeling. Despite a scarcity of studies on interventions for immune remodeling, therapies targeting immune remodeling are promising.
In this section, we will draw from data, following five dimensions to briefly summarize the therapeutic potential of immunomodulation: 1) regular exercise; 2) anti-inflammatory therapy; 3) inflammatory cytokines-targeted therapy; 4) immune cells-targeted therapy, and 5) upstream regulation of immune remodeling.
Exercise has undeniable impacts on the immune (Duggal et al., 2019) and cardiovascular systems (Meissner et al., 2011). Regular physical exercise at low-moderate intensity is recommended as a feasible non-pharmacological therapy for AF patients (Chung et al., 2020). However, the underlying mechanistic detail remains unclear but can be attributed to the modulation of immune, fibrosis, and vagal tones (Guasch et al., 2013;Aschar-Sobbi et al., 2015). It has been suggested that regular voluntary physical activity alters the proliferation of hematopoietic stem and progenitor cells via modulation of their niche and reduces the inflammatory leukocyte output (Frodermann et al., 2019). Monocytes and neutrophils are extremely important elements for atrial inflammation and other risk factors for AF. However, a study suggested that exercise might have a dichotomous effect on the immune system in populations carrying a high burden of AF risk factors. High-intensity and long-term physical training resulted in increased leukocyte output and pro-inflammatory cytokine (TNF-α, IL-2, IL-6 and IL-8) release (Santos et al., 2007;Kawanishi et al., 2015;de Barcellos et al., 2021), along with a greater susceptibility to AF (Guasch et al., 2013;Aschar-Sobbi et al., 2015). Recently, Valenzuela and his colleagues performed a meta-analysis with 6 (n = 935,742) and 4 (n = 2,422) studies to analyze the association of AF with physical activity or sports practice, respectively. Their results suggested that physical activity was overall inversely associated with incident AF whereas high-intensity physical training in athletes was associated with a higher risk for AF (Valenzuela et al., 2022). Of note, there is not yet a strong evidence expounding the association of sports with AF.
Inflammatory responses are the most evident feature of immune remodeling. The widely available, low-cost, and anti-inflammatory drug, colchicine, was once thought to be have preventive potential for postoperative AF. In a sub-study of the COPPS trial, colchicine 1 mg twice daily on the third postoperative day, followed by 0.5 mg twice daily for a month, lowered the incidence of postoperative AF at 30 days compared to placebo (Imazio et al., 2011). Deftereos et al. also showed that colchicine administration, at a dose of 0.5 mg twice daily, for 3 months after pulmonary vein isolation in patients with paroxysmal AF, resulted in a significantly lower rate of AF recurrence over a median of 15 months of follow-up (Deftereos et al., 2014). However, a recent randomized controlled trial (1 mg of colchicine 24 h before the surgery, as well as on days 2, 3, 4, and 5 in the postoperative period) did not detect any statistically significant differences between the control and colchicine groups within 7 days after surgery (Shvartz et al., 2022). Two other studies also found similar results (Bessissow et al., 2018;Tabbalat et al., 2020). Although the above-mentioned opposite outcomes may be attributed to the small sample size and early study termination, the effectiveness of colchicine in preventing AF needs to re-examination. The release of cytokines/-chemokines is an important modulation of the immune system. Targeted treatment of specific cytokines or receptors has attracted considerable academic interest. In rats with sterile pericarditis, treatment with anti-IL-17A monoclonal antibodies markedly alleviated inflammation Frontiers in Physiology frontiersin.org and fibrosis and suppressed the development of AF (Fu et al., 2015). Zhang et al. (2020b) also showed that targeting the CXCL-1/CXCR2 signaling could prevent and reverse the development of AF in spontaneously hypertensive rats. Trials in this research direction have already been conducted in clinical settings. A large, randomized clinical trial involving 10,061 patients with previous myocardial infarction demonstrated that canakinumab targeting of IL-1β significantly lowered the risk of recurrent cardiovascular events as compared to placebo (Ridker et al., 2017). Another trial in patients with peAF showed that canakinumab administration after electrical cardioversion could lower the incidence of AF recurrence at six months; however, no significant differences were observed due to the limited sample size (Krisai et al., 2020). Thus, the effectiveness of these drugs warrants further evaluation in larger multicenter randomized clinical trials. Immune cells constitute the main factors for immune remodeling in AF. Targeted cell therapy may provide an effective strategy. Sun et al. (2016) suggested that the depletion of macrophages can relieve LPS-induced atrial electrical remodeling and AF vulnerability in mice. Wang et al. (2019) also confirmed the effects of depleting macrophages on AF induction. However, prior studies have showed that macrophages played an indispensable role in cardiac homeostasis. Thus, this approach remains controversial (Hulsmans et al., 2017). In addition, a recent study employed engineered CD8 + T cells as a therapeutic agent for treating cardiac fibrosis. Adoptively transferred antigen-special CD8 + T cells could target the fibroblast activation protein expressed on cardiac fibroblasts, thereby suppressing cardiac fibrosis (Aghajanian et al., 2019). This approach can be attempted in future investigations on AF treatment. Finally, immune remodeling may be modulated by a complex combination of physical and neuro-humoral factors in AF. LSG ablation has been shown to reduce macrophage infiltration in the atria and vulnerability to AF after an acute stroke . Therefore, treatment regimens targeting the upstream modulators of immune remodeling can be very efficient. However, the majority of the data derived from animal experiments. Due to differences among species in several aspects, especially the immune system, there is still a long way to verifying the efficacy and achieving the translation from animal experiments to clinical settings.

Conclusion and future prospects
During AF, changes in the composition and number of immune cells, as well as in the levels of immune molecules, constitute immune remodeling, which is inextricably linked with atrial electrical, structural and neural remodeling. Recent attempts to prevent AF by modulating immune remodeling have also suggested the important role of immunity in AF. However, the complexity of immune cell subtypes and the heterogeneity of existing research also make the translation from current data to clinical practice both promising and challenging. Overall, this area of research is only beginning to evolve, and in-depth studies are still needed.

Author contributions
QZ provided the idea and designed the outline. YY reviewed the literatures, drafted the manuscript and designed the figures. MY and DL were involved in refining and polishing the manuscript. All authors contributed to the article and approved the submitted version.

Funding
This work was supported by the National Natural Science Foundation of China (no. 81970277 to QZ).